Summary auto-generated
This research describes the characterization of a carbapenem antibiotic regulatory system in Serratia marcescens, a human pathogen. Unlike Erwinia carotovora, which produces the same carbapenem antibiotic (Car) only in late-exponential growth phase in response to the pheromone OHHL (N-(3-oxohexanoyl)-L-homoserine lactone), S. marcescens constitutively produces carbapenem throughout growth without apparent pheromone involvement. The researchers isolated cosmids from an S. marcescens gene library that complemented E. carotovora carbapenem mutants. These contained a homologue of the carR gene, encoding a LuxR-family transcriptional regulator. The S. marcescens CarR protein shows 59% amino acid identity to E. carotovora CarR and contains conserved DNA-binding and potential autoinducer-binding domains. Critically, S. marcescens CarR functions independently of OHHL, as demonstrated by its ability to restore carbapenem production to an E. carotovora carR carI double mutant without OHHL addition. The protein was expressed and confirmed at approximately 28 kDa. These findings reveal that despite sequence conservation with pheromone-dependent regulators, the S. marcescens CarR can activate carbapenem biosynthesis constitutively, explaining the continuous antibiotic production phenotype observed in this organism.
Key findings
- S. marcescens produces carbapenem constitutively throughout growth, unlike E. carotovora which requires OHHL pheromone signaling
- The S. marcescens carR gene encodes a LuxR-family regulator (CarR) that functions independently of OHHL despite maintaining conserved protein domains
- S. marcescens CarR can functionally replace E. carotovora CarR in trans, restoring wild-type carbapenem levels to E. carotovora carR carI double mutants without exogenous OHHL
- The S. marcescens CarR protein (28 kDa) shows 59% amino acid identity to E. carotovora CarR and retains conserved DNA-binding and putative autoinducer-binding domains despite OHHL-independence
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Abstract
Strain ATCC 39006 of Serratia marcescens makes the same carbapenem, (5R)-carbapen-2-em-3-carboxylic acid (Car), as the Erwinia carotovora strain GS101. Unlike E. carotovora, where the onset of production occurs in the late-exponential phase of growth in response to the accumulation of the small diffusible pheromone N-(3-oxohexanoyl)-L- homoserine lactone (OHHL), in S. marcescens carbapenem is produced throughout the growth phase and does not appear to involve any diffusible pheromone molecule. Two cosmids capable of restoring antibiotic production in E. carotovora group I carbapenem mutants were isolated from an S. marcescens gene library. These cosmids were shown to contain a homologue of the E. carotovora carR gene, encoding a CarR protein with homology to the LuxR family of transcriptional regulators. The S. marcescens carR was subcloned and shown to be capable of complementing in trans, in the absence of OHHL, an E. carotovora carR carI double mutant, releasing the heterologous E. carotovora host from pheromone dependence for carbapenem production. The apparent OHHL- independence of the S. marcescens CarR explains the constitutive nature of carbapenem production in this strain of S. marcescens.